Structure–function analyses reveal the molecular architecture and neutralization mechanism of a bacterial HEPN–MNT toxin–antitoxin system

Abstract

Toxin-antitoxin (TA) loci in bacteria are small genetic modules that regulate various cellular activities, including cell growth and death. The two-gene module encoding a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain and a cognate MNT (minimal nucleotidyltransferase) domain have been predicted to represent a novel type II TA system prevalent in archaea and bacteria. However, the neutralization mechanism and cellular targets of the TA family remain unclear. The toxin SO_3166 having a HEPN domain and its cognate antitoxin SO_3165 with an MNT domain constitute a typical type II TA system that regulates cell motility and confers plasmid stability in the bacterium Here, we report the crystal structure and solution conformation of the SO_3166-SO_3165 pair, representing the first complex structures in this TA family. The structures revealed that SO_3165 and SO_3166 form a tight heterooctamer (at a 2:6 ratio), an organization that is very rare in other TA systems. We also observed that SO_3166 dimerization enables the formation of a deep cleft at the HEPN-domain interface harboring a composite R4-6H active site that functions as an RNA-cleaving RNase. SO_3165 bound SO_3166 mainly through its two α-helices (α2 and α4), functioning as molecular recognition elements. Moreover, their insertion into the SO_3166 cleft sterically blocked the R4-6H site or narrowed the cleft to inhibit RNA substrate binding. Structure-based mutagenesis confirmed the important roles of these α-helices in SO_3166 binding and inhibition. Our structure-function analysis provides first insights into the neutralization mechanism of the HEPN-MNT TA family.

The Variety in the Common Theme of Translation Inhibition by Type II Toxin–Antitoxin Systems

“…Structures are colored in rainbow from N-terminus (blue) to C-terminus (red), in cases of dimers the second monomer is in gray. Structures were visualized using ChimeraX, PDB codes were following: E. coli MazF:3nfc; E. coli HicAB:6hpb (Manav et al, 2019); S. oneidensis SO_3166-SO3165 (HEPN): 5yep (Jia et al, 2018); H. pylori HP0315 (VapD): 3ui3 (Kwon et al, 2012); S. flexneri VapC D7A : 5ecw (Xu et al, 2016); E. coli RelE R81A,R83A : 2kc9 (Li et al, 2009); E. coli HipA S150A : 3tpb (Schumacher et al, 2012); E. coli AtaT Y 144F : 6gtp (Jurenas et al, 2019); prophage P1 Phd-Doc: 3k33 (Garcia-Pino et al, 2010). In the cases of structures in complexes with antitoxin, the coordinates of antitoxin were deleted.…”

“…The SO_3166 toxin cleaves mRNA, but not rRNA or tRNA in vitro, however, the sequence specificity has not been determined. The toxin has a typical all-alpha helical HEPN fold and conserved Arg-(4-6X)-His motif, and forms a dimer with a potential composite active site in a central cleft that could accommodate RNA substrate (Figure 3) (Jia et al, 2018).…”

It takes two (Las1 HEPN Endoribonuclease Motifs) to cut the RNA right

“…1C) (26). This is reminiscent to all characterized HEPN nucleases, which require dimerization of two HEPN domains to activate nuclease activity (4,5,7,27,28). Furthermore, the composite Las1 HEPN active site resembles that of other HEPN nucleases, which are formed by the juxtaposition of two RfxxxH HEPN motifs (Fig.…”

It takes two (Las1 HEPN endoribonuclease domains) to cut RNA correctly

“…1C) (26). This is reminiscent to all characterized HEPN nucleases, which require dimerization of two HEPN domains to activate nuclease activity (4,5,7,(26)(27)(28). Furthermore, the composite Las1 HEPN active site resembles that of other HEPN nucleases, which are formed by the juxtaposition of two RfxxxH HEPN motifs (Fig.…”

“…The expanding list of HEPN nucleases highlights their profound role in numerous biological processes, most notably host-defense and stress response pathways. For example, the eukaryotic HEPN nuclease Ire1 triggers the unfolded protein response and the bacterial HEPN nuclease SO_3166 is the toxic component of the dueling type II toxin-antitoxin system (4,5). In contrast to these defense systems, the HEPN nuclease Las1 ensures the translational capacity of the cell through its role in ribosome assembly (6).…”